While high breast density, as measured by mammography, is one of the strongest predictors of breast cancer risk, we know little about the biological basis of breast density or why or how it is associated with increased cancer risk. To address this problem we need a combined clinical/ basic science approach to obtain more accurate and informative techniques to measure mammographic density in the clinic, a fuller understanding of the biology that generates breast density and, most importantly, a way to identify specific aspects of this biology that contribute to increased risk for human breast cancer. Our multidisciplinary investigation will first and foremost examine human tissue to address the histologic and pathophysiologic basis of breast density. To extend these studies, we will use animal models to test predictions generated by the analysis of human tissues since murine models provide an in vivo setting that can be more easily manipulated. We will use (and develop) high-resolution, volumetric bio-imaging to spatially co-register clinical X-ray images of breast density to the histology and tissue composition that underlies breast density. This information will guide our molecular analysis of the same tissue to produce a comprehensive molecular and cellular portrait of breast density. Using a powerful combination of in vivo and in vitro structural, genetic, molecular and functional analyses of human tissue, we will identify candidate markers that link high breast density with an increased risk for breast cancer. We will use large, well-defined and unique population-based cohorts to test our hypotheses and validate markers that would enhance a clinician's ability to identify those women at significantly increased risk for breast cancer. We hypothesize that increased breast density may be the end result of biologic processes that result in altered cell-cell and/or cell-extracelluar matrix (ECM) interactions and that these are causal for increased breast cancer risk. These altered interactions are influenced by genetic, physiologic and environmental factors and generate the tissue phenotypes that are characteristic of high breast density (excess collagen, tissue remodeling, etc.). These phenotypic characteristics have been observed in conditions where response to tissue remodeling or damage is occurring such as in mammary gland development (branching morphogenesis), wound healing or the desmoplastic reaction of malignancies. In these processes, "activated stroma" results in increased levels of collagen and tenascin, stromal remodeling and altered cell cycle control for cellular components located within. Prior work from two of our Investigators (Tlsty and Barcellos-Hoff) has shown that such stroma can dramatically influence tumorigenesis in both human and murine models. Proper stromal-epithelial interactions can actually suppress the expression of preneoplastic phenotypes in epithelial cells and conversely, altered stromal-epithelial interactions can promote the probability that preneoplastic lesions progress to malignancy. The combination of information from the novel imaging in Project 1, the cellular, molecular and functional analyses in Projects 2 and 4 and the epidemiological assessment of molecular markers in Project 3 has the potential to create several new and clinically useful, radiographic and/or molecular measures of breast density that are more specific than mammographic density for estimating cancer risk.